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Identification of Sex Hormone Binding Globulin-Interacting Proteins in the Brain Using Phage Display Screening

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Identification of Sex Hormone Binding Globulin-Interacting Proteins in the Brain Using Phage Display Screening Gnanasekar 16/04 14/8/2009 12:23 ÌÌ Page 421 INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 24: 421-426, 2009 421 Identification of sex hormone binding globulin-interacting proteins in the brain using phage display screening M. GNANASEKAR1, F.G. SULEMAN2, K. RAMASWAMY1 and J.D. CALDWELL3 1Department of Biomedical Sciences, University of Illinois College of Medicine, 1601 Parkview Avenue, Rockford, IL 61107; 2Thermo Fisher Scientific, 3747 N. Meridian Road, Rockford, IL 61105; 3Department of Pharmacology, Lake Erie College of Osteopathic Medicine, 1858 West Grandview Boulevard, Erie, PA 16509, USA Received April 16, 2009; Accepted June 2, 2009 DOI: 10.3892/ijmm_00000248 Abstract. The present study reports the identification of Nakhla et al (2,3) demonstrated that SHBG binds to a receptor human sex hormone binding globulin (SHBG)-interacting or receptor complex which then results in increases of intra- proteins in the brain using a phage display-based screening cellular cAMP. Studies by Rosner and colleagues (3,4) suggest technology. Phage display is a system in which a foreign that the putative SHBG receptor is a G protein-coupled receptor protein is displayed on the surface of a bacteriophage as a or functionally linked to one. In these studies (1) a 167-kDa fusion protein with one of the coat proteins of the bacteriophage. protein was solubilized from membranes of human prostate T7 phage clones expressing normal human brain proteins glands and its binding activity to SHBG was demonstrated (human normal brain phage-display cDNA expression library) using 125I-SHBG. Other studies (5,6) also suggested that there were screened using SHBG as bait. The bound phage clones are membrane-associated receptors for SHBG in the uterus. were then identified by DNA sequencing and by BLAST These membrane-associated receptors may be important for search analysis. Of the twenty binding proteins analyzed, three internalization of SHBG, which was observed in many tissues were found to be membrane-associated proteins: synaptosomal (7-14) including the brain (15). The Nykjaer and Willnow associated protein 25 (SNAP25), Thy-1 cell surface antigen and laboratory demonstrated that a membrane-associated protein zonadhesin. Further studies will determine if the interactions of called megalin, a 600-kDa member of the LDL receptor gene SHBG with these proteins have any role in the internalization family, is capable of binding to and facilitating the internalization and cell signaling events or whether they contribute to steroid of both SHBG and its bound steroid (16). It is the protein that delivery to specific cells. the same laboratory previously suggested was involved in the uptake of other steroids and demonstrated that it belonged to Introduction a larger class of proteins responsible for the uptake of a broad range of lipophilic molecules (17). Megalin is important in Examining the roles of steroid binding globulins as active steroid effects. This group demonstrated that without megalin, participants in steroid actions has received attention recently. mice showed a delayed onset of puberty (16). The same One reason is that there are membrane-associated receptors laboratory demonstrated the importance of binding globulins for binding globulins. Hryb et al (1) showed that sex hormone in mediating the effects of steroids. Mice lacking a cortico- binding globulin (SHBG) binds with high affinity to a specific steroid-binding globulin, another steroid-binding globulin membrane receptor in prostate stromal and epithelial cells. found in the brain (18,19), had more free steroids in the blood but showed fewer physiological responses to them, suggesting that the presence of this binding globulin was necessary in _________________________________________ order to see the effects of corticosteroids (20). The first indication that there were SHBG receptors in the Correspondence to: Dr Munirathinam Gnanasekar, Department brain was when SHBG facilitated female sexual receptivity (21) of Biomedical Sciences, University of Illinois College of Medicine, except when coupled to dihydrotestosterone (22). Our laboratory 1601 Parkview Avenue, Rockford, IL 61107, USA and others also found that SHBG is produced in the brain, E-mail: [email protected] where it is often co-localized with the neuropeptides oxytocin Dr Jack D. Caldwell, Department of Pharmacology, Lake Erie and vasopressin (23-25). Since both SHBG and oxytocin are College of Osteopathic Medicine, 1858 West Grandview Boulevard, often found together in synaptic vesicles in axonal varicosities, Erie, PA 16509, USA we concluded that SHBG is likely released into the brain. E-mail: [email protected] This suggests the existence of receptors for this released SHBG. We also presented indirect evidence of such SHBG Key words: sex hormone binding globulin, brain, phage display, receptors in the hypothalamus (26). After binding to membrane- zonadhesin, synaptosomal associated protein 25 associated receptors, it is possible that SHBG, its associated steroid, and the SHBG receptor are all internalized into brain cells. Caldwell et al (15) demonstrated uptake of SHBG in vivo Gnanasekar 16/04 14/8/2009 12:23 ÌÌ Page 422 422 GNANASEKAR et al: SHBG-INTERACTING PROTEINS Figure 2. Size distribution of phage clones obtained from first round of biopanning. Phage bound to SHBG from the first cycle of biopanning were eluted and amplified on BLT5403. The amplified phages were plated with BLT5403 on LB agar plates for plaque formation. Ten plaques were randomly selected for PCR amplification. Amplified PCR products (5 μl) were loaded on 1% agarose gel containing Etbr and electrophoresed for 30 min. The resolved gel was observed on a UV transilluminator and the image was captured using Kodak digital software. Lane 1, control PCR that lacks a Figure 1. Schematic strategy to identify targets of SHBG. template; lanes 2-11 random phage clones. in the choroids plexus, paraventricular and supraoptic nuclei, and in specific periventricular cells. Despite observations that of the boiled samples were used as DNA templates for PCR suggest the presence of both central and peripheral SHBG amplification. membrane receptors, the identity of brain SHBG receptors has not been elucidated. Another study utilized a yeast two-hybrid PCR and DNA sequencing. DNAs of the selected phage clones system to identify candidate proteins for SHBG binding in were amplified by PCR according to the manufacturer's prostate (27). In this study, phage display of a human brain (Novagen) instructions on a Perkin Elmer Thermocycler cDNA library is used to identify SHBG-interacting proteins. using the T7Select up 5'-GGAGCTGTCGTATTCCAGTC-3' and T7Select down 5'-GGCTGATACCACCCTTCAAG-3' as Materials and methods forward and reverse primers respectively. PCR conditions were 30 cycles of denaturation at 94˚C for 30 sec, 55˚C annealing Materials. The normal human brain T7Select™ phage library, for 30 sec, and 72˚C extension for 30 sec. A final extension at host strain BLT5403, as well as primers were obtained from 72˚C was for 5 min. The reaction products were resolved on Novagen, USA. Qiaquick PCR purification kits were from agarose gel to confirm the presence of amplification products. Qiagen (Valencia, CA). Jumpstart TAQ polymerase was from The PCR products were then purified using the Qiaquick Sigma (St. Louis, MO). PCR purification kit (Qiagen, Valencia, CA) and the inserts were sequenced on both strands at DNA Services Facility Biopanning. The strategy to identify the SHBG interacting (Research Resources Center, University of Illinois at Chicago, proteins is presented in Fig. 1. Briefly, biopanning was IL) to confirm the authenticity of the genes. The determined performed as follows. SHBG was diluted in deionized water to DNA sequences were then analyzed by BLAST analyses. 10 μg/ml; 100 μl aliquots were coated in the wells of a 96-well microtiter plate (Costar, Corning, NY) and incubated at room Results temperature (RT) for 3 h. The SHBG-coated wells were then washed with Tris-buffered saline (TBS), blocked by adding Over twenty phage clones bound to SHBG. Of these, initially 200 μl 3% BSA to each well and incubated at RT for 1 h. we isolated ten phage plaques from the fourth round of bio- Excess blocking reagent was removed by washing with panning and processed them for sequencing. Agarose gel deionized water. The T7Select phage of the human normal resolution of the PCR products showed that they were all the brain cDNA library (Novagen, USA) was added to wells and same size (data not shown), so we sequenced two clones out of incubated at RT for 1 h. Unbound phages were washed off the ten. Sequence analysis demonstrated that SHBG-interacting with TBS containing Tween while bound phages were then phage clones encoded for FAU gene products [ubiquitin-like eluted by incubating each well with 200 μl of 1% SDS in protein (FUBI) and ribosomal protein S30] (28). TBS. The eluted phages were then used to infect E. coli Because of the low molecular weight of these two candidate BLT5403 which amplifies the phages for the next biopanning proteins, we decided to screen more plaques from the first cycle. Three more biopanning rounds were done to increase biopanning. Through this process we isolated twenty-four the phage binding affinity to the SHBG. Finally, the phages more phage clones. All these clones were then processed for obtained after this fourth amplification were plaque titered sequencing. Fig. 2 shows the size distribution of the phage with a series of dilutions (10-2 to 10-14) on LB agar plates clones that were obtained after PCR amplification. The size containing 1% agarose and incubated for 2-4 h at 37˚C. Single of these clones varied between 570 and 1,265 bases. Each of plaques of phages were randomly selected with a pipette tip, the sequences was then subjected to a BLAST search to resuspended in 100 μl of 0.1X TE buffer, boiled for 10 min determine their identity. Table I shows the list of candidate and placed on ice for 5 min.
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